Ileitis diagnostic assay

ABSTRACT

Improved immunoassays for the protection of antibodies against  Lawsonia intracellularis  are provided which permit rapid, easy detection of low concentrations of anti- Lawsonia  antibodies in animal-derived specimens. The preferred assay is an ELISA assay employing an antigenic extract of  L. intracellularis  lipopolysaccharide.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is broadly concerned with improved immunoassaysfor the detection of antibodies against Lawsonia intracellularis, aswell as an effective antigen comprising and preferably consistingessentially of an antigenic extract of L. intracellularislipopolysaccharide. The preferred assay is an indirect-type ELISA assayhaving excellent specificity and sensitivity, allowing use of the assayfor detection of low levels of antibody in animal-derived specimensduring initial stages of infection prior to the onset of clinical signsof disease.

2. Description of the Prior Art

Porcine proliferative enteritis (PPE), known as ileitis, intestinaladenomatosis, or necrotic enteritis, is a naturally occurring diseasethat can affect pigs from waning to young adult stage. PPE was formerlybelieved to be caused by a camplyobacter-like organism or ileal symbiontintracellularis. More recently, it has been established that thecausative agent is Lawsonia intracellularis, an obligate intracellular,gram-negative bacterium. The disease is of economic importance owing todeath loss, increased medication costs, poor weight gain and decreasedfood conversion in affected animals.

A key element in the rational therapy and effective control of PPE is arapid and accurate identification of etiologic agents. PPE may bediagnosed by observation of gross lesions and is confirmed byobservation of specific hystopathological lesions in which theintracellular curved rods are demonstrated by special staining methodsincorporating the use of an anti-Lawsonia monoclonal antibody. Ideally,a final determination should be made through the isolation of thecausative agent. However, the isolation and culture of L.intracellularis requires specialized cell culture techniques.

Attempts have been made in the past to develop rapid assays for thedetection of anti-Lawsonia antibodies. Current serological-based assayssuch as indirect fluorescence antibody test (IFAT) and immuno-peroxidaseassay (IPMA) demonstrate good sensitivity and specificity in thedetection of anti-Lawsonia antibodies antemortem in pig serum. Knittel JP, Jordan D M, Schwartz J K, et al. Evaluation of Antemorten PolymeraseChain Reaction and Serological Methods for Detection of LawsoniaIntracellularis-exposed Pigs. American Journal of Veterinarian Research.59:722-726 (1998). Guedes R M C, Gebhart C J, Deen J, et al. Validationof an Immunopreoxidase Monolayer Assay as a Serological Test for PorcineProliferative Enteropathy. Journal of Veterinarian DiagnosticInvestigation. 14:528-530(2002), the teachings and content of thesereferences are hereby incorporated by reference. However, neither ofthese assays is sensitive enough to detect the lower concentrations ofanti-Lawsonia antibodies often found in pig serum during the initialexposure and onset of infection time periods. In addition, these priorassays rely on highly skilled technicians to accurately conduct thetests and interpret the results, e.g., the results are subjectivelyobtained by spending many hours looking into a microscope, analyzingwells illuminated by UV or natural light, searching for L.intracellularis or L. intracellularis-infected cells stained fluorescentgreen or red representing a “positive” sample.

Enzyme-linked immunoassays (ELISA) have also been developed fordetection of anti-Lawsonia antibodies. These prior efforts failed toproduce a sufficiently sensitive and specific assay owing to variouslimitations including poor antigen quality and quantity, variability inantibody titers, overlapping antibody titer between infected andnon-infected pigs lack of a valid positive/negative “cut-off” value, andcross-reactivity of pig antibodies to non-specific antigen components.Holyoake P K, Cutler R S, Caple I W, Monckton R P. Enzyme-linkedImmunosorbent Assay for Measuring Ileal SymbiontIntracellularis-specific Immunoglobulin G Response in Sera in Pigs.Journal of Clinical Microbiology. 31: 1980-1985 (1994), the teachingsand content of which is hereby incorporated by reference.

There is accordingly a need in the art for an improved anti-Lawsoniaantibody assay which is highly sensitive and specific, permittingaccurate detection of low concentrations of antibodies in animal-derivedspecimens during the early stages of infection.

Lipopolysaccharides (LPS) are a major suprastructure of gram-negativebacteria such as L. intracellularis which contributes greatly to thestructural integrity of the bacteria, and protects them from host immunedefenses. LPS's form a part of the Gram negative bacteria cell wall andcomprise three parts: polysaccharide side chains, core polysaccharidesand lipid A. Lipid A may contain unusual fatty acids (e.g.,hydroxy-mysteric acids) while core polysaccharides often contain unusualsugars (e.g., KDO, keto-deoxyoctulonate and heptulose). Thepolysaccharide side chain is referred to as the O-antigen of thebacteria. LPS's function as endotoxin, because they can bind to the CD14receptor of macrophage, triggering the whole cascade formacrophage/endothelial cells to secrete pro-inflammatory cytokines.

SUMMARY OF THE INVENTION

The present invention overcomes the problems outlined above and providesan improved immunoassay for detecting the presence of antibodies againstLawsonia intracellularis with a high degree of specificity andsensitivity, allowing the assay to be used for the early detection ofPPE. Broadly speaking, the assay of the invention is an immunoassaywherein an animal-derived specimen is contacted with an effective amountof an antigen comprising at least a portion of a lipopolysaccharide ofL. intracellularis, causing the formation of complexes between theantigen and the antibodies, and then determining the presence of suchantibodies. In preferred forms, the assay is an indirect ELISA test.

The animal-derived specimen is most preferably sera, but can alsoinclude colostrums, joint fluids, salivas, tissue homogenates and feces.These specimens can be prepared in accordance with conventionaltechniques for assay purposes. Although the invention is particularlyconcerned with detection of PPE in swine, analogous Lawsonia-causeddiseases can also be detected in animals such as pigs, hamsters, bluefox, emus, deer, dogs, guinea pigs, horses, rhesus macaque monkeys,ostriches, rabbits and rats. The antibodies detected using the methodsof the invention generally are selected from the group consisting ofIgG, IgA and IgM antibodies.

The preferred antigen for use in the immunoassays of the invention is aportion or extract of a lipopolysaccharide of L. intracellularis. Thisextract preferably has a molecular weight of from about 15-25 kDa, morepreferably from about 18-20 kDa. The extract or portions thereof shouldbe of sufficient size and antigenicity to induce an immune response inthe animal upon administration of an effective amount of the antigen.Furthermore, the extract or portion thereof must be of sufficient sizefor antibody-antigen complexes with LPS antibodies. In particularlypreferred forms, the LPS antigen exhibits an endotoxicity of from about3-75 EU/ml (more preferably from about 10-60 EU/ml, still morepreferably from about 20-50, and even more preferably from about 25-40EU/ml) using the bacteria endotoxin test. The antigen preferablyconsists essentially of LPS abtract, and good results have been obtainedwith an extract of L. intracellularis ATCC Accession No. PTA-4927,deposited on Jan. 9, 2003, with the ATCC, 10801 University Blvd.,Manassas Va. 20110-2209. PTA-4927 was tested by the ATCC on Apr. 21,2004 and was found to be viable. The date of conversion to the BudapestTreaty was Oct. 16, 2007.

The most preferred ELISA is a indirect-type assay and involves firstcoating the wells of a microtiter plate with LPS, followed by overnightincubation at room temperature. A blocking agent is then added withfurther overnight incubation at 4° C. The selected dilute detectionantibody is then added with incubation at 37° C. for one hour, followedby diluted conjugate with further one hour (37° C.) incubation. Next,the TMB chromagen is added with incubation at room temperature for fiveminutes. At this point the reaction is stopped with the addition of 2Msulfuric acid and the plate is read at 450 nm.

The preferred ELISA test is optimized in the case of a starting LPSextract sample of ATCC Accession No. PTA-4927 having an endotoxin levelof about 34.75 EU/ml. In such an instance, the antigen should be presentat a dilution of from about 1:250 to 1:8000, more preferably from about1:400 to 1:6000, still more preferably from about 1:500 to 1:4000, evenmore preverably from about 1:600 to 1:3000, still more preferably fromabout 1:750 to 1:2000, even more preferably from about 1:900 to 1:1500,with 1:1,000 being optimum. The detection antibody should be present ata dilution level of about 1:20-1:320, 1:25-1:240, 1:30-1:128, 1:35-1:60with 1:40 being the best. The ELISA conjugate should be used at a levelof from about 1:250-1:2000, 1:300-1:1500, 1:350-1:1000, 1:400-1:600,with 1:500 being preferred. Of course, appropriate dilution levels maybe readily calculated if the starting antigen has a different endotoxinlevel than the preferred product of the invention.

The present invention also lends itself to recombinant LPS antigensbeing used as the antigenic source. In such instances, the LPS antigenor portion thereof will be generated using conventional recombinanttechniques. For example, DNA encoding for the LPS antigen or desiredportion thereof can be inserted expression invectors and operably linkedto expression control sequences which then permit the expression vectorto express the desired LPS antigen or portion thereof. The expressedantigen is then recovered and used in accordance with the presentinvention. Of course, those of skill in the art are familiar withvarious ways of producing and recovering recombinant antigens inaccordance with the present invention. In all instances, recombinantantigens in accordance with the present invention will preferentiallybind to or hybridize with L. intracellularis antibodies.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following examples set forth presently preferred techniques forconstructing and using Lawsonia LPS as an antigenic source forimmunoassays. Such examples include an indirect-type ELISA immunoassayagainst Lawsonia antibodies. It is to be understood, however, that suchexamples are provided by a way of illustration only, and nothing thereinshould be construed as a limitation upon the overall scope of theinvention.

EXAMPLE I Development and Confirmation of LPS-Based Indirect ELISA Assay

Bacterial Antigen Preparation

The bacterial isolate was identified as high passage (>20 passages pastinitial isolation from the affected gut) L. intracellularis isolate#15540 (ATTC Accession No. PTA-4927). This isolate was acquired from aDanish sow affected with acute hemorrhagic proliferative enteropathy(HPE), as confirmed by routine histology and immunohistochemistry (IHC)staining techniques and co-cultured to obtain a pure culture of L.intracellularis by methods previously described. Lawson G H K, McOristS, Jasni, et al. Intracellular Bacteria of Porcine ProliferativeEnteropathy: Cultivation and Maintenance in Vitro. Journal of ClinicalMicrobiology. 31:1136-1142 (1993), the teachings and content of whichare incorporated by reference herein. Multiple 30 L batches of L.intracellularis #15540 were propagated using fresh McCoy cell (ATCC#1696) suspensions in bioreactors (Applicon, Inc., Foster City, Calif.).Active cultures were allowed to reach 80-100% cell infectivity and thenharvested by centrifugation using an Avanti Beckman J-20I centrifuge(Beckman Instruments, Inc., Fullerton, Calif.), JA-10 rotor at 17,000×gfor 15 minutes at 4° C. The supernatants of each batch were discardedand cell pellets containing both extracellular L. intracellularis andMcCoy cells infected with L. intracellularis were resuspended in 30 mlsterile 1× phosphate-buffered saline (PBS) and stored at −80° C.

In order to purify L. intracellularis from McCoy cells, a discontinuouspercoll gradient was prepared following methods previously describedwith slight modifications. Holyoake P K, Cutler R S, Caple I W, MoncktonR P. Enzyme-linked Immunosorbent Assay for Measuring Ileal SymbiontIntracellularis-specific Immunoglobulin G Response in Sera in Pigs.Journal of Clinical Microbiology. 31:1980-1985 (1994), the teachings andcontent of which are hereby incorporated by reference. Briefly, 225 mlof percoll (Amersham Biosciences, Pharmacia Biotech, Uppsala, Sweden)was mixed with 260 ml of reagent grade (RG) water and 15 ml of 5M sodiumchloride (NaCl, Fisher Brand). Harvested cultures mentioned above werepassed >20 times through a 25 gauge needle and 5 ml of bacterial/McCoycell homogenate was transferred to 25 ml of the percoll gradient into 30ml polycarbonate centrifuge tubes. Tubes were mixed by inversion andcentrifuged at 37,000×g for 1 h at 4° C. The ensuing suspensioncontained scattered cellular debris in the upper 50% of the tube whileone distinct cellular banding pattern was visualized at a buoyantdensity of 1.075 g/ml. The upper half of the gradients were removedwhile the bands were carefully collected using a 5 ml polypropylenepipette and transferred to new 30 ml centrifuge tubes containing 20 mlof sterile PBS. The tubes were centrifuged (Avanti Beckman J-20I, JA-17rotor) at 37,000×g for 15 minutes at 4° C. and the process repeated amaximum of 3 times to wash out the percoll from each sample. After thefinal centrifugation step, the pellets were resuspended in 1 to 2 ml ofsterile PBS, pooled, aliquoted into 1.8 ml cryovial tubes (Nalgene,Nalgene Nunc Int'l., Rochester, N.Y.) and stored at −80° C. A sample ofthe resuspension containing highly concentrated, percoll purified L.intracellularis #15540 was observed under dark field microscopy toconfirm presence of tiny curved rods and absence of intact McCoy cells.The LPS antigenic component was extracted from the percoll purified L.intracellularis #15540 antigen with hot aqueous phenol using methodspreviously described with slight modifications. Westphal, O. andLuderitz, O. Chemische Erforschung von Lipopolysacchariden GramnegativerBacterien. Angew. Chemical 66: 407-17 (1954), the content and teachingof which are hereby incorporated by reference. Briefly, 18 ml of percollpurified L. intracellularis and 3.75 ml of phenol chloroform (Ameresco,Solon, Ohio), pH 8.0 was incubated separately in a 65° C. water bath for10 minutes. After the initial incubation period, 4.5 ml of the percollpurified L. intracellularis was transferred to each tube containing0.9375 ml of hot phenol chloroform and gently mixed by inversion. Tubeswere incubated for an additional 25 minutes in the 65° C. water bath,mixing by inversion every 5 minutes during incubation and then cooledovernight at 4° C. Slight phase separation of aqueous and solid phasesoccurred in each tube during cold storage. Each tube was centrifuged(Avanti Beckman J-20I, JA-17 rotor) at 7,700×g for 25 minutes at 4° C.and the LPS-containing supernatant from all 4 tubes was pooled andretained while the cell pellets were discarded. The supernatant wastransferred to pre-sterilized dialysis tubing (Spectrum Laboratories,Inc., Rancho Domingo, Calif.), placed in a 4 L plastic beaker anddialyzed in cold reagent grade (reverse osmosis) water for 24 to 48hours to remove the phenol chloroform from the sample. The reagent gradewater was exchanged with fresh water every 2 to 4 hours until thewashing step was complete. The resultant purified L. intracellularis LPSextract was carefully collected and stored at −80° C. until use.

Confirmation of the LPS extract was visualized through separation on4-12% Bis-Tris pre-cast gel (NuPAGE, Invitrogen, Carlsbad, Calif.) bysodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) inMOPS running buffer (NuPAGE, Invitrogen). The LPS extract was comparedto percoll purified whole-cell L. intracellularis and uninfectedwhole-cell McCoy cells. Samples were prepared for SDS-PAGE by dilutingeach 1:2 into 4× lithium salt dodecyl sulfate (LDS) denaturing buffer(NuPAGE, Invitrogen) and incubated in a 85° C. water bath for 10minutes. The gel was periodate silver stained following proceduresoutlined by Bio-Rad Silver Stain instructions with modifications. Inbrief, the gel was subjected to a primary fixation in 40% Methanol(MeOH, Fisher Brand, Hanover Park, Ill.)/10% acetic acid (Fisher Brand)(v/v) for 20 to 30 minutes followed by incubation in 40% MeOH/10% aceticacid (v/v) containing 0.7% periodic acid for an additional 5 minutes atroom temperature. A secondary fixation involved transferring the gelinto 10% ethanol (EtOH)/5% acetic acid (v/v) for 5 minutes followed byoxidation in oxidizer reagent (Bio-Rad, Hercules, Calif.) for 5 minutesthen, washed in deionized water until the yellow color was gone from thegel. The gel was incubated in 0.16 mM dithiothreitol (DTT) in water for5 minutes and stained in silver reagent (Bio-Rad) for 20 minutes at Roomtemperature. The gel was washed once in deionized water for 20 to 40seconds and placed in developing reagent (Bio-Rad) until the desiredstrength of banding appeared. The reaction was stopped in 5% (v/v)acetic acid for 20 minutes at room temperature and washed once indeionized water. Proteins were identified by their molecular mass usinga 10 to 220 kDa protein marker (BenchMark, Invitrogen).

LPS-Based Indirect ELISA

Anti-Lawsonia immunoglobulin G (IgG) antibodies were detected in testsamples using an L. intracellularis LPS-based indirect enzyme linkedimmunosorbent assay (LPS-ELISA). Lawsonia intracellularis LPS was coatedat 100 μl/well on Immulon 2HB plates (Dynex, Chantilly, Va.) at a 1:1000dilution in 0.05M sodium carbonate coating buffer, pH 9.6, sealed withmylar plate sealers (Thermo Labsystems, Franklin, Mass.) and incubatedat Room temperature for 24 hours. Each plate was washed using theUltrawash PLUS (Dynex), 350 ml/well, zero soak time, for 1 wash cyclewith wash buffer containing 0.05% Tween 20 (Fisher Brand), 0.137M NaCl(Fisher Brand), 0.005M potassium chloride (KCl, Sigma, St. Louis, Mo.),0.009M Sodium Phosphate, diabasic (Na₂HPO₄, Sigma), 0.001M potassiumphosphate (KH₂PO₄, Sigma), pH 7.2 to 7.4, in RG water. Antigen-coatedplates were blocked at 300 ml/well with blocking buffer containing 5%(v/v) non-fat dried milk (Bio-Rad) in SeaBlock™ (Pierce Biotech,Rockford, Ill., containing steelhead salmon serum and 0.1% sodium oxidein PBS) at 4° C. for 24 hours to prevent non-specific binding of testsera to the plates. Each plate was then washed for 3 cycles as mentionedabove. Test sera and controls were pre-diluted 1:40 in blocking buffer.

Fifty microliters per well of diluted sera was transferred to each platein duplicate, sealed and incubated at 37° C. for 1 hour. The washingsteps were repeated for 3 cycles before adding 50 μl/well of a 1:500dilution of goat anti-swine IgG horseradish peroxidase (HRP) conjugate(Kirkegaard and Perry Laboratories, Inc., Gaithersburg, Md.) in blockingbuffer. Plates were sealed and incubated at 37° C. for 1 hour. Thewashing steps were repeated for 3 more cycles and then, 50 μl/well ofperoxidase substrate consisting of a 2-component 3,3′,5,5′tetramethylbenzidine (TMB, KPL) was applied to each plate and incubatedat Room temperature for 5 minutes. This was done to observe the presenceof antigen-antibody complexes within each test sample. The colorimetricreaction was stopped by transferring 50 μl/well of 2M sulfuric acid(H₂SO₄, Fisher Brand) solution. Plates were read at 450 nm wavelength ona V-max 96-well microtiter plate reader (Medtechs, Inc., Chapel Hill,N.C.) to obtain optical density (OD) values for each test sample.Standard controls containing a range of high positive to low positive(by serial two-fold dilution of the highly positive sample in blockingbuffer) and negative antibodies against the L. intracellularis LPSextract were included in each plate. The test plates were consideredvalid if the coefficient of determination (r²-value) was ³ 0.9 in alinear regression analysis of the standard's OD values. An empty wellnot containing test or control samples served as a blank control for alltests.

Validation of LPS-ELISA

Porcine sera were obtained from the following studies for use invalidating the LPS-ELISA for detection of anti-Lawsonia IgG antibodiesin pigs. (i) Two pigs approximately 3 weeks of age were hyperimmunizedby intramuscular injection with 2 ml of L. intracellularis LPS extractformulated 1:2 with Freund's incomplete adjuvant (Sigma). One pigapproximately 3 weeks of age was given a 2 ml intramuscular injection ofplacebo consisting of Dulbecco's Minimal Essential Medium (DMEM) with 5%(v/v) bovine serum (JRH Biosciences, Lenexa, Kans.) formulated 1:2 withFreund's incomplete adjuvant. Boosters of each inoculum wereadministered 3 weeks and 6 weeks post initial inoculation for generationof positive (antibodies to L. intracellularis LPS) and negative (no L.intracellularis antibodies) control test serum. Four to six millilitersof serum was collected from each pig prior to inoculation, at 3 weeksand 6 weeks post inoculation and tested for reaction of L.intracellularis whole cell antigen by an indirect florescent antibodyserology test (IFAT), Knittel J P, Jordan D M, Schwartz J K, et al.Evaluation of Antemorten Polymerase Chain Reaction and Serologic Methodsfor Detection of Lawsonia Intracellularis-exposed Pigs. American Journalof Veterinarian Research. 59:722-726 (1998), the teachings and contentof which are hereby incorporated by reference, to confirm presence orabsence of anti-Lawsonia antibodies in serum samples. At 8 weekspost-initial inoculation, animals were euthanised and a final serumcollection was obtained. The positive control serum from each pig waspooled, tested to confirm positive reaction to L. intracellularis byIFAT, and both anti-Lawsonia LPS positive and negative control serum wasstored in 1 ml aliquots at −80° C.

Porcine sera from 2 previously conducted vaccine efficacy studies,Kroll, J., et al. (2004). Evaluation of protective immunity in pigsfollowing oral administration of an avirulent live vaccine of Lawsoniaintracellularis. AJVR 65(5): 559-565, the teaching and content of whichis hereby incorporated by reference, were tested to investigate ananti-Lawsonia LPS antibody positive/negative optical density cut-offlimit. Test serum from eighty 6 to 9 week old pigs previously confirmedto be IFAT-positive for anti-Lawsonia antibodies were generated afterexperimental infection with a virulent heterologous L. intracellularisisolate N101494 (Boehringer Ingelheim Vetmedica, Inc., St. Joseph, Mo.).Test serum previously confirmed to be IFAT-negative, was collected fromeighty 3 to 9 week old strict control pigs that did not receive avaccination or challenge any time during each clinical study.

175 serum samples were collected from 25 pigs after vaccination with alive attenuated L. intracellularis vaccine, Enterisol® Ileitis(Boehringer Ingelheim Vetmedica, Inc.), after challenge with a virulentheterologous L. intracellularis isolate N101494 or both. Another 70serum samples were collected from 10 strict control pigs that did notreceive a vaccination or challenge and remained IFAT negative foranti-Lawsonia antibodies throughout the study. The study design includedthirty five 3 to 4 week old pigs randomly blocked into 3 treatmentgroups. On day 0 of the study, 15 pigs from group 1 received a 2 ml oraldose of vaccine while groups 2 and 3 (10 pigs/group) received anequivalent dose of placebo consisting of uninfected McCoy cellsuspension in growth medium. On day 21, pigs in groups 1 and 2 weregiven an intragastric dose of virulent heterologous pure culturechallenge of L. intracellularis N101494. On day 42, pigs were necropsiedand evaluated for lesion development to identify efficacy of thevaccinated pigs compared to non-vaccinated, challenged pigs. Fecalsamples and serum were collected weekly from day 0 to 42 for routinediagnostic testing to detect rates of exposure and active shedding of L.intracellularis due to vaccine or challenge administration. Lesions wereevaluated to confirm presence of PE at day 42 only by gross examination,histological and IHC methods as described below.

Confirmation of PE in Pigs

Gross lesions found in the ileum or colon of pigs in clinical studiesdescribed above were scored according to the severity of mucosalthickness (1=normal, 2=mild thickening, 3=moderatethickening/inflammation, 4=severe thickening/inflammation/mucosalhemorrhaging or necrosis may be present). Kroll, J., et al. (2004).Evaluation of protective immunity in pigs following oral administrationof an avirulent live vaccine of Lawsonia intracellularis. AJVR 65(5):559-565. Samples 2-4 cm in length of ileum and colon were collected postmortem, fixed by immersion in buffered formalin and processed fordetection of microscopic lesions. This included Hematoxylin and Eosin(H&E) and IHC staining incorporating specific L. intracellularismonoclonal antibodies. Kroll, J., et al., AJVR, (2004). The latter isconsidered the current standard for assessment of the actual infectionstatus of a pig with L. intracellularis. Kroll, J., et al. (2004).Microscopic lesions found in IHC stained tissue were scored separatelyaccording to severity of L. intracellularis specific cell proliferation(0=normal, 1=mild/focal, 2=moderate/diffuse, 3=severe/diffuse). Averagegross and microscopic lesion scores and the frequency of lesionsdetected in the affected tissue were calculated for group comparisons.Average gross and macroscopic lesion scores were considered the primaryparameter for determining vaccine efficacy against virulent heterologouschallenge in previous studies. Kroll, J., et al. (2004).

EXAMPLE II

Preferred LPS-ELISA Materials and Methods

The following sets forth the presently preferred LPS-ELISA assay inaccordance with the invention.

A. Protocol

1. Materials

-   -   a. LPS Binding Plates:        -   Immunlon 2 HB 96 well plates, Dynex Cat. No. 3455 or            equivalent.    -   b. Dilution Plates:        -   Falcon Pro-Bind Assay Plate (Fisher Scientific, Pittsburg,            Pa.), 96-well, U-Bottom without lid (polystyrene,            non-sterile), Becton Dickinson (San Diego, Calif.) Cat. No.            353910 or equivalent.    -   c. Plate Sealers:        -   Mylar Plate Sealer, Thermo Labsystems (Franklin, Mass.) Cat.            No. 5701 or equivalent.    -   d. Coating Buffer:        -   0.05M Sodium Carbonate buffer            -   10.6 g Na₂CO₃ Sigma Cat. No. S6139 or equivalent.            -   QS with reagent grade (RG) H₂O (or equivalent) to 1.0 L.            -   pH=9.6±0.1            -   Store at 2-7° C. until use.            -   Expiry: 7 days.    -   e. Wash Solution:        -   0.05% Tween 20, 0.137M NaCl, 0.005M KCl, 0.009M Na₂HPO₄,            0.001M KH₂PO₄            -   32.0 g NaCl.            -   0.8 g KCl.            -   2.44 g Na₂HPO₄.            -   0.8 g KH₂PO₄.            -   QS with RG H₂O or equivalent to 4.0 L.            -   pH to 7.2-7.4 with NaOH or HCl.            -   2.0 ml of Tween, Fisher Cat No. BP337-100 or equivalent.            -   Store at room temperature (25±5° C.) until use.            -   Expiry: 1 week.    -   f. Blocking Solution:        -   5% Non-fat dry milk in Seablock™.            -   25.0 g Non-fat dry milk. Bio-Rad Cat No. 170-6404 or                equivalent.            -   QS to 500 mL with Seablock™. Pierce Biotech Cat. No.                37527 or equivalent.            -   Store at 2-7° C. until use.            -   Expiry: 1 month.    -   g. Antigen:        -   1:1000 dilution of lipopolysaccaride whole molecules from L.            intracellularis.            -   40 μl L. intracellularis LPS into 40 ml of coating                buffer.            -   Use immediately.    -   h. Detection Antibody:        -   1:40 dilution of convalescent pig serum antibodies to L.            intracellularis.            -   3 μl pig serum or equivalent into 120 μl of blocking                solution.            -   Store at 2-7° C. until use.            -   Expiry: 24 hours.    -   i. Conjugate antibody:        -   1:500 dilution of Goat anti-mouse IgG (H+L)—Horse Radish            Peroxidase (HRP). Kierkegaard and Perry Laboratories, Inc.            Cat. No. 14-14-06 or equivalent.            -   40 μl conjugate into 20 ml of blocking solution.            -   Store at 2-7° C. until use.            -   Expiry: 24 hours.    -   j. Substrate:        -   Two-Component Microwell Peroxidase Substrate (Gaithersburg,            Md.). KPL Cat No. 50-76-00 or equivalent.            -   Mix equal volumes of TMB Peroxidase Substrate                (Reagent A) with Peroxidase Solution B (Reagent B)                immediately prior to use.            -   Volume required=5 mL/plate. Therefore, 2.5 mL of Reagent                A+2.5 mL of Reagent B for 1 test plate.            -   Store at 2-7° C. until use.            -   Expiry: Pre-mixed reagents per manufacturer's suggested                expiration date. Use mixed substrate solution                immediately.    -   k. Stop Solution:        -   2M H₂SO₄.            -   In a fume hood, carefully mix: 444.4 mL of RG H₂O.            -   55.6 mL of 18M H₂SO₄ Fisher Cat No. A300c-212, or                equivalent.            -   Store at room temperature until use.            -   Expiry: 6 months.    -   l. Positive Control        -   1:2,564 dilution of hyperimmunized pig serum containing            anti-Lawsonia LPS IgG antibodies.            -   3.9 μl of positive control Lot #090203 into 10 ml of                blocking solution            -   Store at 2-7° C. until use.            -   Expiry: 24 hours    -   m. Negative Control        -   1:2,564 dilution of hyperimmunized pig serum containing no            antibodies against L. intracellularis LPS molecules.            -   3.9 μl of positive control Lot #090203 into 10 ml of                blocking solution            -   Store at 2-7° C. until use.            -   Expiry: 24 hours

2. Methods

-   -   a. Samples are run in duplicate. Number of plates needed=Total        number of samples/40 samples per plate. Round up to a whole        number of plates. Columns 11 and 12 will contain 1:10 serial        dilutions of negative and positive control serum.    -   b. Dilute L. intracellularis LPS antigen 1:1000 or appropriate        working dilution in coating buffer. Volume required=Number of        plates×10 ml/plate.    -   c. Add 100 ml of diluted antigen to every well of each plate.    -   d. Seal plates with plate sealers and incubate at room        temperature overnight (14-24 hours).    -   e. Wash plates with wash solution using Dynex Ultrawash PLUS,        350 ml/well, zero soak time, for 1 wash cycle. Tap plates dry on        paper towels.    -   f. Add 300 ml of block solution to all wells. Seal plates and        incubate at 2-7° C. overnight (14-24 hours).    -   g. Wash plates with wash solution using Dynex Ultrawash PLUS,        350 μl/well, zero soak time, for 3 wash cycles. Tap plates dry        on paper towels.    -   h. In a U-bottom dilution plate, add 120 μl of blocking solution        sample to wells in columns 1-10 and wells B-H in columns 11 and        12.    -   i. Add 240 μl of negative and positive controls in wells of row        A in columns 11 and 12 respectively.    -   j. Make 10-fold dilutions of each control serum by transferring        120 μl of diluted control in wells A-11 and A-12 to wells B-11        and B-12 using a 50-300 μl multi-channel pipette, taking care        not to transfer diluted negative control to the last well of        column 11 (well H-11) as this sample will serve as a plate        blank.    -   k. Dilute detection antibody (convalescent pig sera) 1:40 in        blocking solution by transferring 3 μl of sample into 120 μl of        blocking solution in each well of the dilution plate. Dilute by        adding a different sample each time to wells A-1, B-1, C-1, etc.    -   l. Using a 50-300 μl multichannel pipette, mix the contents in        column 1 by pipetting up and down at least 3 times and transfer        50 μl/well to columns 1 and 2 of the antigen coated LPS-binding        test plates. Change tips and repeat step until all diluted        samples have been transferred in duplicate across the plate.    -   m. Transfer 50 μl/well of the negative control (wells A-H,        column 11) to corresponding wells of the test plate(s). Repeat        step for the positive control. Each control contains enough        diluted sample to use in 2 test plates.    -   n. Seal test plate(s) with plate sealers and incubate for 1.0        hour±15 minutes at 37° C.±2.0° C.    -   o. Wash plates with wash solution using Dynex Ultrawash PLUS,        350 μl/well, zero soak time, for 3 wash cycles. Tap plates dry        on paper towels.    -   p. Add 50 μl of conjugate antibody diluted 1:500 or appropriate        working dilution to all wells of the test plate(s). Volume        required=Number of plates×5 ml/plate.    -   q. Seal test plate(s) with plate sealers and incubate for 1.0        hour±15 minutes at 37° C.±2.0° C.    -   r. Wash plates with wash solution using Dynex Ultrawash PLUS,        350 μl/well, zero soak time, for 3 wash cycles. Tap plates dry        on paper towels.    -   s. Add 50 μl of substrate to use to all wells of the test        plate(s), incubate at room temperature for five minutes±1        minute.    -   t. Stop the reaction with the addition of 50 μl of Stop solution        to all wells five minutes after the addition of substrate.    -   u. Read plates on a plate reader equipped with a 450 nm        wavelength filter.

3. Interpretation of Results

-   -   a. Test samples exhibiting >0.200 optical density at 450 nm        wavelength are considered positive for anti-Lawsonia LPS IgG        antibodies.    -   b. Test samples exhibiting <0.200 optical density at 450 nm        wavelength are considered negative for anti-Lawsonia LPS IgG        antibodies.

1. An immunoassay for detecting the presence of antibodies against Lawsonia intracellularis in an animal-derived specimen comprising the steps of contacting said specimen with an effective amount of an antigen comprising at least a portion of an isolated lipopolysaccharide of Lawsonia intracellularis, said portion being of sufficient size to form antibody-antigen complexes with Lawsonia intracellularis LPS antibodies and having an endotoxicity of about 3-75 EU/ml, causing the formation of complexes between said antigen and said antibodies, and determining the presence of said complexes, wherein the presence of said complexes indicates the presence of said antibodies.
 2. The immunoassay of claim 1, said specimen selected from the group consisting of animal-derived sera, colostrums, joint fluids, salivas, tissue homogenates, and feces.
 3. The immunoassay of claim 1, said animal selected from the group consisting of pigs, hamsters, blue fox, emus, deer, dogs, guinea pigs, horses, rhesus macaque monkeys, ostriches, rabbits and rats.
 4. The immunoassay of claim 1, said antibodies being selected from the group consisting of IgG, IgA and IgM antibodies.
 5. The immunoassay of claim 1, said immunoassay being an ELISA test.
 6. The immunoassay of claim 5, said antigen being recombinantly derived.
 7. The immunoassay of claim 5, said ELISA test including the steps of immobilizing said antigen on a surface, contacting said specimen with said immobilized antigen, causing a complex between antibodies in said specimen and said antigen, and detecting said complexes.
 8. The immunoassay of claim 1, said lipopolysaccharide having a molecular weight of from about 15-25 kDa.
 9. The immunoassay of claim 8, said molecular weight being from about 18-20 kDa.
 10. The immunoassay of claim 1, said antigen capable of inducing an immune response in said animal upon administration of an effective amount of the antigen to the animal.
 11. The immunoassay of claim 1, said endotoxicity being from about 25-40 EU/ml.
 12. The immunoassay of claim 1, said antigen comprising an isolated lipopolysaccharide extract from the strain deposited as ATCC Accession No. PTA-4927.
 13. An antigen for detecting the presence of antibodies against Lawsonia intracellularis, said antigen comprising at least a portion of an isolated Lawsonia intracellularis lipopolysaccharide, said portion being sufficient size to form antibody-antigen complexes with Lawsonia intracellularis LPS antibodies and having an endotoxicity of about 3-75 EU/ml.
 14. The antigen of claim 13, said lipopolysaccharide having a molecular weight of from about 15-25 kDa.
 15. The antigen of claim 14, said molecular weight being from about 18-20 kDa.
 16. The antigen of claim 14, said antigen capable of inducing an immune response in said animal upon administration of an effective amount of the antigen to the animal.
 17. The antigen of claim 14, said lipopolysaccharide being recombinantly derived.
 18. The antigen of claim 14, said endotoxicity being from about 25-40 EU/ml.
 19. The antigen of claim 13, said antigen comprising an isolated lipopolysaccharide extract from the strain deposited as ATCC Accession No. PTA-4927.
 20. In an enzyme-linked immunoassay for detecting the presence of antibodies against Lawsonia intracellularis in an animal-derived specimen, the improvement which comprises employing an isolated lipopolysaccharide of Lawsonia intracellularis having an endotoxicity of about 3-75 EU/ml, as an antigen in said immunoassay.
 21. The immunoassay of claim 20, said specimen selected from the group consisting of animal-derived sera, colostrums, joint fluids, salivas, tissue homogenates, and feces.
 22. The immunoassay of claim 20, said animal selected from the group consisting of pigs, hamsters, blue fox, emus, deer, dogs, guinea pigs, horses, rhesus macaque monkeys, ostriches, rabbits and rats.
 23. The immunoassay of claim 20, said antibodies being selected from the group consisting of IgG, IgA and IgM antibodies.
 24. The immunoassay of claim 20, said ELISA test being an indirect-ELISA test.
 25. The immunoassay of claim 24, said ELISA test including the steps of immobilizing said antigen on a surface, contacting said specimen with said immobilized antigen, causing a complex between antibodies in said specimen and said antigen, and detecting said complexes.
 26. The immunoassay of claim 20, said lipopolysaccharide having a molecular weight of from about 15-25 kDa.
 27. The immunoassay of claim 26, said molecular weight being from about 18-20 kDa.
 28. The immunoassay of claim 20, said antigen capable of inducing an immune response in said animal upon administration of an effective amount of the antigen to the animal.
 29. The immunoassay of claim 20, said endotoxicity being from about 25-40 EU/ml.
 30. The immunoassay of claim 20, said antigen comprising an isolated lipopolysaccharide extract from the strain deposited as ATCC Accession No. PTA-4927. 